5. Formulation and Development of Microemulsion and SMEDDS

Transcription

1 5. Formulation and Development of Microemulsion and SMEDDS

2 Contents 5 Formulation and Development of Microemulsion and SMEDDS Formulation techniques for Microemulsion Phase titration method (Water titration method): Phase inversion method: Method developed by Boycott and Schulman: Selection of excipients used for microemulsion and SMEDDS Drug solubility determination in oils, surfactants & co-surfactants Drug selected surfactants compatibility study: Optimization of surfactant: co-surfactant ratio by pseudo-ternary phase diagram Microemulsion System: SMEDDS Effect of Drug loading on the phase diagrams of the selected systems Felodipine Microemulsion: Valsartan SMEDDS Preparation of Drug Loaded Microemulsions and SMEDDS Felodipine Microemulsion: Valsartan SMEDDS: References Akshay R. Koli 139

3 List of Tables Table : Solubility of Felodipine in excipients Table : Solubility of Valsartan in excipients Table : Drug - selected surfactant compatibility study for Felodipine Table : Drug - selected surfactants compatibility study for Valsartan Table : Water titration Reading for Phase diagram (Microemulsion System) Table : Water titration Readings for Phase Diagram (V1) Table : Water titration Readings for Phase Diagram (V2) Table : Water titration Readings for Phase Diagram (V3) Table : Compositions of Felodipine Microemulsion Systems (Batch F1 F9) Table : Compositions of Valsartan SMEDDS 1 (V1) Table : Compositions of Valsartan SMEDDS 2 (V2) Table : Compositions of Valsartan SMEDDS 3 (V3) Akshay R. Koli 140

4 List of Figures Figure :Pseudoternary phase diagram of oil, water and surfactant mixture showing microemulsion region Figure : Solubility of Felodipine in excipients Figure : Solubility of Valsartan in excipients Figure : Pseudo-ternary phase diagrams of Capmul MCM (oil), Tween20: PEG 400 (S:CoS) and Water system Figure : Excipients profiles for three different systems of SMEDDS Figure : Pseudo-ternary phase diagrams of Capmul MCM (oil), Tween 80: PEG 400 (S:CoS) and Water system Figure : Pseudo-ternary phase diagrams of Capmul MCM (oil), Labrasol: Transcutol P (S:CoS) and Water system Figure : Pseudo-ternary phase diagrams of Capmul MCM (oil), Tween 80: Transcutol P (S:CoS) and Water system Figure : Pseudoternary phase diagram of Capmul MCM, Tween 20 and PEG 400 (Placebo) Figure : Pseudoternary phase diagram of Felodipine, Capmul MCM, Tween 20 and PEG Figure : Pseudoternary phase diagram of Capmul MCM, Tween 80 and PEG 400 (Placebo) Figure : Pseudoternary phase diagram of Valsartan, Capmul MCM, Tween 80 and PEG Figure : Flow Chart of preparation of SMEDDS and Microemulsion Akshay R. Koli 141

5 5 Formulation and Development of Microemulsion and SMEDDS 5.1 Formulation techniques for Microemulsion Many researchers in various literatures have reported the formulation techniques for microemulsion. These techniques include: Phase titration method (Water titration method): Microemulsions are prepared by the spontaneous emulsification method (phase titration method) and can be depicted with the help of phase diagrams. Construction of phase diagram is a useful approach to study the complex series of interactions that can occur when different components are mixed. Microemulsions are formed along with various association structures (including emulsion, micelles, lamellar, hexagonal, cubic, and various gels and oily dispersion) depending on the chemical composition and concentration of each component. The understanding of their phase equilibria and demarcation of the phase boundaries are essential aspects of the study. As quaternary phase diagram (four component system) is time consuming and difficult to interpret, pseudo ternary phase diagram is often constructed to find the different zones including microemulsion zone, in which each corner of the diagram represents 100% of the particular component as shown in Figure They can be separated into w/o or o/w microemulsion by simply considering the composition that is whether it is oil rich or water rich. Observations should be made carefully so that the metastable systems are not included [1]. In this method, at a constant ratio of S/CoS, various combinations of oil and S/CoS are produced and the water is added drop wise. After the addition of each drop, the mixture is stirred and examined through a polarized filter or by naked eye. The appearance (transparency, opalescence and isotropy) is recorded after addition of each drop of water [2]. Akshay R. Koli 142

6 Figure :Pseudoternary phase diagram of oil, water and surfactant mixture showing microemulsion region Phase inversion method: Phase inversion of microemulsions occurs upon addition of excess of the dispersed phase or in response to temperature. During phase inversion drastic physical changes occur including changes in particle size that can affect drug release both in vivo and in vitro. These methods make use of changing the spontaneous curvature of the surfactant. For non-ionic surfactants, this can be achieved by changing the temperature of the system, forcing a transition from an o/w microemulsion at low temperatures to a w/o microemulsion at higher temperatures (transitional phase inversion). During cooling, the system crosses a point of zero spontaneous curvature and minimal surface tension, promoting the formation of finely dispersed oil droplets. This method is referred to as phase inversion temperature (PIT) method. Instead of the temperature, other parameters such as salt concentration or ph value may be considered as well instead of the temperature alone. Additionally, a transition in the spontaneous radius of curvature can be obtained by changing the water volume fraction. By successively adding water into oil, initially water droplets are formed in a continuous oil phase. Increasing the water volume fraction changes the spontaneous curvature of the surfactant from initially stabilizing a w/o microemulsion to an o/w microemulsion at the inversion locus. Short-chain surfactants form flexible monolayers at the o/w interface resulting in a bicontinuous microemulsion at the inversion point [1]. Akshay R. Koli 143

7 5.1.3 Method developed by Boycott and Schulman: In this method, adding the oil, surfactant mixture to some of the aqueous phase in a temperature controlled container with agitation makes a coarse macro emulsion as a first step, which is then titrated with co-surfactant until clarity is obtained and then diluted with water to give a microemulsion of the desired concentration [2]. The desired characteristics of microemulsions and SMEDDS are dilutability, transparency, globule size in the range of 100 nm for enhanced absorption, zeta potential around -10 to -30 mv for stability [3]. The parameters which can affect these properties are the nature and concentration of oil, surfactant and co-surfactant. The ratio of surfactant: co-surfactant plays a very important role in successful preparation of microemulsion. Hence these parameters were studied and optimized to obtain the desirable microemulsion formulation. The dependent parameters were Dilutability, Percentage transmittance, Droplet size and Zeta potential [4]. Here water titration method was used for preparation of microemulsion and SMEDDS because it is easy & scalable. Akshay R. Koli 144

8 5.2 Selection of excipients used for microemulsion and SMEDDS Development of microemulsion systems for poorly water soluble drugs is critical. Components selected for the formulation should have the ability to solubilize the drug in high level to deliver the therapeutic dose of the drug in an encapsulated form. In general, excipients with higher solubilizing efficiency for drug are selected for formulation development Drug solubility determination in oils, surfactants & co-surfactants Solubility of drugs was determined in different oils (such as capmul MCM, Capryol 90, Capmul MCM C8, Capmul MCM C10, Captex 200P, Captex 355, Isopropyl myristate, Soyabean oil, Castor oil), surfactants (such as Tween 20, Tween 80, Labrasol, Plurol oleique, Cremophore EL) and co-surfactants (such as Transcutol P, PEG 400, Labrafil 1944 CS). Non-ionic surfactants were used in this study since they are known to be less affected by ph and changes in ionic strength. Drug was added in excess amount into 2 ml of each component in vials and stirred for 48 hrs at 25 C on magnetic stirrer. The mixture vials were then kept at 25±1.0 0 C in an isothermal shaker for 72 h to reach equilibrium [6, 7]. The equilibrated samples were removed from shaker and centrifuged at 3000 rpm for 15 min to remove the excess drug, after which the concentration of drug in supernatant was measured by UV spectrophotometric method after appropriate dilution with methanol. Then drug solubility (mg/ml) was calculated and depicted in Table and for Felodipine and Valsartan respectively. Result and Discussion The components used in the system should have high solubilization capacity for the drug, ensuring the solubilization of the drug in the resultant dispersion. The higher solubility of the drug in the oil phase is important for the microemulsion and SMEDDS to maintain the drug in solubilized form. In present study, oils namely capmul MCM, capmul MCM C8, capmul MCM C10, captex 200P, captex 355, castor oil, isopropyl myristate and olive oil were screened for solubilization of both drugs. Akshay R. Koli 145

9 In the present study, non ionic surfactants namely tween20, tween 80, labrasol, peceol and plurol oleique were screened as nonionic surfactants are less toxic than ionic surfactants. Microemulsion dosage forms for oral or parenteral use based on nonionic surfactants are likely to offer in vivo stability [8]. Transient negative interfacial tension and fluid interfacial film is rarely achieved by the use of single surfactant, usually necessitating the addition of a co-surfactant. The presence of co-surfactant decreases the bending stress of interface and allows the interfacial film sufficient flexibility to take up different curvatures required to form microemulsion over a wide range of composition. Thus, the co-surfactants namely PEG 400, propylene glycol and Transcutol P were screened for the study that again are nonionic surfactants. Since the Felodipine and Valsartan are highly lipophilic, it was presumed that keeping them in lipophilic environment might increase their stability [9]. Felodipine: The solubility of Felodipine in different oils, surfactants, co-surfactants and water was determined (Table and Figure ). The solubility of Felodipine was found to be highest in Capmul MCM (90±1.25 mg/ml) as compared to other oils while in water it was mg/ml [10]. This may be attributed to the polarity of the poorly water soluble drugs that favor their solubilization in small/medium molecular volume oils such as medium chain triglycerides or mono- or diglycerides [11]. Akshay R. Koli 146

10 Table : Solubility of Felodipine in excipients Ingredients Oils Solubility (mg/ml) Capmul MCM 90 ± 1.25 Capmul MCM C8 80 ± 1.17 Capmul MCM C10 30 ± 2.57 Captex 200P ± 3.09 Captex ± 2.36 Castor oil 8.50 ± 4.24 Olive oil 3.87 ± 2.40 Isopropyl myristate 2.78 ± 1.44 Surfactants Tween ± 2.52 Tween ± 2.47 Labrasol 15 ± 1.25 Plurol oleique 10 ± 3.30 Co-surfactants PEG ± 3.44 Propylene glycol Insoluble Akshay R. Koli 147

11 Solubility (mg/ml) Excipients Figure : Solubility of Felodipine in excipients The studies have revealed that mixed mono and diglyceride like Capmul gave microemulsion (clear or translucent liquid) and emulsion phases, whereas di- and triglycerides exhibited an additional gel phase. Among individual mono-, di- and triglycerides, the oil-in-water microemulsion region was found to be the largest for the diglyceride. Dispersion of drug in aqueous media from mixtures of mono- and diglyceride or mono- and triglyceride was superior to individual lipids [12]. Monodiglyceride medium chain esters like Capmul MCM are particularly recommended for the dissolution of difficult compounds [13]. Hence Capmul MCM was selected as the oil phase. The solubility of Felodipine was also very high in Tween 20 and PEG 400. Hence these components were selected as surfactant and co-surfactant for microemulsion system preparation. Valsartan: The solubility of Valsartan in different oils, surfactants, co-surfactants and water was determined (Table and Figure ). The solubility of Valsartan was found to Akshay R. Koli 148

12 be highest in Capmul MCM (110±1 mg/ml) as compared to other oils while in water it was 0.003±0.01mg/ml. This may be attributed to the polarity of the poorly water soluble drugs that favor their solubilization in small/medium molecular volume oils such as medium chain triglycerides or mono- or diglycerides [11]. Table : Solubility of Valsartan in excipients Ingredients Solubility (mg/ml) Oils Capmul MCM 110 ± 1.2 Capmul MCM C10 74 ± 6.6 Capmul MCM C8 70 ± 1.6 Captex 200 P 15 ± 3.06 Captex 355 NF 42 ± 3.5 Olive oil 8 ± 3.5 Isopropyl myristate 10 ± 1.5 Surfactants Tween ± 5.5 Labrasol 90± 1.2 Peceol 82± 3.00 Co-surfactants PEG ± 2.7 Transcutol P 12 ± 0.5 Akshay R. Koli 149

13 Solubility (mg/ml) 5. Formulation and Development of Microemulsion and SMEDDS Excipients Figure : Solubility of Valsartan in excipients From above data, highest solubility of Valsartan was found in capmul MCM as oil. Due to suitability of Capmul MCM as an oil phase as per previous discussion, it was selected as oil phase. All three surfactants i.e. Tween 80, Peceol and Labrasol show comparable solubility of Valsartan. Thus all of them were taken for further studies. The solubility of Valsartan was almost similar in PEG 400 and Transcutol P. Thus Tween 80, peceol and Labrasol were selected as surfactants and PEG 400 and Transcutol P as co-surfactants for SMEDDS preparation Drug selected surfactants compatibility study: Physical compatibility of the water-insoluble drug with surfactants should be used in surfactant selection procedure. Physical compatibility may include precipitation/crystallization, phase separation and color change in the drug surfactant solution during course study. Chemical compatibility is primarily regarded as the chemical stability of the drug in a surfactant solution. A surfactant was considered for further development only if it was physically and chemically compatible with drug. A Akshay R. Koli 150

14 fixed amount (5 ml) of each of the surfactant:co-surfactant (1:1) was placed in a 10 ml glass vial with a known amount (100 mg) of drug. The samples were stored under 25 o C for 1 month and observed for physical changes and analyzed for chemical changes [14]. Results and Discussion The drug and surfactant compatibility study was designed to evaluate the effect of Tween 20 on the physical and chemical stability of Felodipine and effect of Tween 80, Peceol and Labrasol on the physical and chemical stability of Valsartan. This study was found to be very useful because concentrations of surfactants are usually quite high in microemulsion formulations. As data demonstrated in Table and , there were no significant losses of potency (less than 10%) in any of the samples. Felodipine did not show any signs of incompatibility with surfactant and co-surfactant mixture. The results are as shown in Table Table : Drug - selected surfactant compatibility study for Felodipine Surfactant : Co-Surfactant Mixture (1:1) Tween 20:PEG 400 Precipitation Crystallization Phase separation Color change % Recovery(1 month at 25 0 C) 99.0 Where, - Presence and - Absence In case of Valsartan, Peceol : PEG 400 and Labrasol : PEG 400 combinations showed precipitation during 1 month study. Thus they were eliminated from further studies. The remaining S:CoS combinations passed the Durg-Surfactant compatibility test. These results showed promise for a SMEDDS formulation which could be the way to proceed further to meet the dose requirement for Valsartan. The results are as shown in Table Akshay R. Koli 151

15 Table : Drug - selected surfactants compatibility study for Valsartan Surfactant:CoSurfac tant Mixture (1:1) Precipitati on Crystallizati Akshay R. Koli 152 on Phase separati on Color chan ge % Recovery (1 month at 25 0 C) Tween 80:PEG Labrasol : Transcutol P 97.8 Peceol : PEG Tween 80: Transcutol P 99.3 Labrasol : PEG Where, - Presence and - Absence 5.3 Optimization of surfactant: co-surfactant ratio by pseudo-ternary phase diagram The existence of microemulsions regions were determined using pseudo-ternary phase diagrams. The mixture of oil and surfactant/co-surfactant at certain weight ratios were diluted with water in a drop wise manner. Distill water was used as an aqueous phase for the construction of phase diagrams. For construction of pseudo ternary phase diagrams, water titration method was used because this method is easy & scalable. In this study, microemulsions were prepared to find the area of particular component system [6, 15, 16]. In this method, surfactant was blended with co-surfactant in fixed weight ratios i.e. 1:1, 2:1, 3:1, and 4:1 for Felodipine Microemulsion and 3:1, 2:1 and 1:1 for Valsartan SMEDDS. As from reports, it was found that at S/CoS (0.5/1) stable microemulsion formation is not possible. Aliquots of each surfactant and co-surfactant mixture (S mix ) were then mixed with oil at ambient temperature. For each phase diagram, the ratio of oil to the S mix was varied as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 (% v/v). Water was added drop wise to each oil-s mix mixture under vigorous stirring. After equilibrium, the

16 samples were visually checked and determined as being clear microemulsion or emulsion or gel. No heating is conducted during the preparation. These values of oil, surfactant and co-surfactant were used to determine the boundaries of microemulsion region [17]. After the identification of microemulsion region in the phase diagrams, the microemulsion formulations were selected at desired Surfactant : Co-surfactant (Smix) ratios. To determine the effect of drug addition in SMEDDS, phase diagrams were constructed in presence of drug. Black color shows self-microemulsion region and gray color indicates microemulsion region. In order to prepare SMEDDS, selection of microemulsion region from phase diagram was based on the fact that solution remains clear even on infinite dilution [6, 15, 16]. Phase diagrams were prepared using Pro-Sim ternary diagram software. Results of phase diagram system are shown in Table for Felodipine and Table to for Valsartan. Results and Discussion Pseudo-ternary phase diagrams were constructed to identify the Microemulsifying regions. It has been observed that increasing concentration of the Surfactant within the microemulsifying region caused increased spontaneity of self micro-emulsification process. When a CoS was added to the system, it further lowered the interfacial tension between the oil and water interface and also influenced the interfacial film curvature and stability. On the other hand, safety should be considered with the increasing concentration of S and CoS. All the combinations under test formed a microemulsion in certain concentrations, but the combination with wider single phase region is considered to be a better combination in terms of microemulsification efficiency Microemulsion System: The system for Felodipine microemulsion is composed of oil (Capmul MCM), surfactant:co-surfactant (Tween 20:PEG 400) and distilled water. Akshay R. Koli 153

17 Table : Water titration Reading for Phase diagram (Microemulsion System) Oil (ml) S mix (ml) Dilution with water until system remained clear (ml) 4:1(S:CoS) 3:1(S:CoS) 2:1(S:CoS) 1:1(S:CoS) infinite infinite Infinite Infinite A B Akshay R. Koli 154

18 C D Figure : Pseudo-ternary phase diagrams of Capmul MCM (oil), Tween20: PEG 400 (S:CoS) and Water system Microemulsion (Single phase region) In microemulsion system, surfactant and co-surfactant get preferentially adsorbed at the interface, reducing the interfacial energy as well as providing a mechanical barrier to coalescence. The decrease in the free energy required for the emulsion formation consequently improves the thermodynamic stability of the microemulsion formulation. Therefore, the selection of oil and surfactant, and the mixing ratio of oil to S/CoS, play an important role in the formation of the microemulsion. This can ascertain by pseudoternary phase diagram as it differentiates the microemulsion region from that of macroemulsion region. The water titration results for phase diagram are shown in Table for Felodipine. One can select the microemulsion region from pseudo-ternary phase diagram. As seen in figure , the microemulsion existence area increased as the concentration of S:CoS ratio increased. The grey region in the phase diagram is the one phase region which is the characteristic of Microemulsion. As shown in Figure (A), for the 4:1 ratio of Tween 20:PEG 400 (S:CoS), more than 40% of S:Cos is required to stabilize 10% of the oil to make a single phase system. The phase diagram shows that when S:CoS reduces less than 35%, coarse Akshay R. Koli 155

19 emulsion forms having particle greater than 100 nm (Fig A). Hence it can be predicted that concentration of S:CoS should be more than 35% to form Microemulsion. Higher concentration of oil leads to turbidity and coarse emulsion system. The decrease in surfactant concentration in the system i.e. 3:1 ratio of S:CoS (Fig B)didn t show any significant difference compared to the previous 4:1 ratio. In figure (Fig (C)), 2:1 ratio of S/CoS covers maximum microemulsion region as compare to other ratio of S/CoS. In this system, 40% of S/CoS can incorporate more than 12% of the oil which is the highest incorporation of oil among all S:CoS ratio. Above these concentrations coarse emulsion formed. When the ratio of S:CoS was 1:1 (Fig (D)), minimum microemulsion region was observed compared to other ratios and showed fairly low incorporation of water to maintain visually clear microemulsion systems. It involves formation of microemulsion which is unstable on dilution after 20% oil. Initially it formed microemulsion but later on converted to emulsion as it moved towards higher concentration of oil. Because of this, they were not selected for further investigation. Hence putting into Nut Shell, 2:1 ratio of S/CoS forms better microemulsion region and more water incorporation to form visually clear microemulsion compared to 1:1 ratio and almost similar to 3:1 and 4:1 ratio and hence selected for further development and in all the cases concentration of oil should be less than 20%v/v SMEDDS Valsartan SMEDDS prepared using three different systems are summarized in Figure considering the solubility study of the drug in various solvents. SMEDDS formed oil in water microemulsion with gentle stirring, upon being introduced into aqueous media. Since the free energy of the microemulsion is very low, the formation is thermodynamically spontaneous. Surfactant and co-surfactant formed a layer around the droplet of microemulsion, which not only reduced the interfacial energy but also provided a mechanical barrier to coalescence. Generally, high proportion of oil in microemulsion may result in high solubilization for poorly water-soluble drugs. However, O/W microemulsions were not formed when SMEDDS with high proportions of oil were diluted. Therefore, only SMEDDS with the law levels of oil were studied. Akshay R. Koli 156

20 SMEDDS ( : Ingredients used) Ingredients V 1 V 2 V 3 Capmul MCM Tween 80 Labrasol PEG 400(CoS) Transcutol P (CoS) Figure : Excipients profiles for three different systems of SMEDDS Table : Water titration Readings for Phase Diagram (V1) V1: Oil (Capmul MCM), Surfactant : co-surfactant (Tween 80:PEG 400) and water. Oil (ml) Smix (ml) Dilution with water until system remain clear (ml) 3:1(S:CoS) 2:1(S:CoS) 1:1(S:CoS) Infinite Infinite Infinite Infinite Infinite Infinite Akshay R. Koli 157

21 Figure : Pseudo-ternary phase diagrams of Capmul MCM (oil), Tween 80: PEG 400 (S:CoS) and Water system Self Microemulsion Microemulsion Akshay R. Koli 158

22 Table : Water titration Readings for Phase Diagram (V2) V2: Oil (Capmul MCM), Surfactant : co surfactant (Labrasol :Transcutol P) and water. Oil (ml) Smix (ml) Dilution with water until system remain clear (ml) 3:1(S:CoS) 2:1(S:CoS) 1:1(S:CoS) Akshay R. Koli 159

23 Figure : Pseudo-ternary phase diagrams of Capmul MCM (oil), Labrasol: Transcutol P (S:CoS) and Water system Self Microemulsion Microemulsion Table : Water titration Readings for Phase Diagram (V3) V3: Oil (Capmul MCM), Surfactant:co surfactant (Tween 80:Transcutol P) and water. Oil (ml) Smix (ml) Dilution with water until system remain clear (ml) 3:1(S:CoS) 2:1(S:CoS) 1:1(S:CoS) Infinite Infinite Infinite Infinite Infinite Akshay R. Koli 160

24 Figure : Pseudo-ternary phase diagrams of Capmul MCM (oil), Tween 80: Transcutol P (S:CoS) and Water system Self Microemulsion Microemulsion Pseudo-ternary phase diagram for each formulation shown above represents presence of microemulsion and emulsion regions. Black region represents self microemulsion domain where as gray region indicates formation of microemulsion. Akshay R. Koli 161

25 SMEDDS forms microemulsion when titrated with water under agitation condition. The particle size of microemulsion is less than 100 nm and as the energy required to form microemulsion is very low, it is a thermodynamically spontaneous process [18]. This process is facilitated by presence of surfactant. The surfactant forms a layer around oil globule in such a way that polar head lies towards aqueous and non polar tail pull out oil and thereby reduces surface tension between oil phase and aqueous phase [20, 21]. Another factor affecting formation of microemulsion is the ratio of surfactant and co-surfactant. The lipid mixtures with different surfactant, co-surfactant and oil ratios lead to the formation of SMEDDS with different properties [22]. Since surfactant and co-surfactant adsorb at interface and providing mechanical barrier to coalescence, selection of oil, surfactant, co-surfactant and mixing ratio to S/CoS, play important role in microemulsion [23, formation 24]. Nine different composition systems were prepared to study pseudoternary phase diagram using surfactants and co-surfactants in varying ratio. Generally, high proportion of oil in microemulsion may result in high solubilization for poorly water-soluble drugs. However, no O/W microemulsion was formed when SMEDDS with high proportion of oil were diluted. System V1 was prepared using Capmul MCM as oil phase, Tween 80 as surfactant and PEG 400 as co-surfactant. Formulation V1 A was prepared with surfactant : co-surfactant (S:CoS) ratio of 3:1. As shown in Figure (A), the point where amount of oil is less than 10%, the water content is around 90%. At this point microemulsion can be diluted to infinite which fulfills requirement of SMEDDS and also particle size of this microemulsion is less than 100nm (described in characterization of SMEDDS). The region where oil content is more than 20% and surfactant: co-surfactant is up to 60% also forms the microemulsion but these were found to be unstable on dilution. The phase diagram shows that when S:CoS reduces less than 40%, coarse emulsion forms having particle greater than 100 nm (Fig (A)). Hence it can be predicted that concentration of Smix should be more than 40% to form self-microemulsion. Further, more amount of oil also entrap less water content and thereby results in coarse emulsion. As shown in Figure (Fig (B)), formulation V1 B covers maximum microemulsion region as compare to all other formulations. Formulation V1B was prepared using similar Akshay R. Koli 162

26 excipients but with S/CoS ratio of 2:1. In this system, after dilution amount of oil contained was limited up to 20% and concentration of S/CoS was also 50%. At this point and below, microemulsion can be diluted to infinite which fulfills requirement of SMEDDS and also particle size of this microemulsion is less than 100nm (described in characterization of SMEDDS). Above these concentrations coarse emulsion formed. The third formulation V1C was prepared using S:CoS as 1:1. Formulation V1 C covers minimum microemulsion region compared to V1 A and B. It involves formation of microemulsion which is unstable on dilution after 20% oil (Fig (C)). Initially it formed self microemulsion but later on converted to emulsion as it moved towards higher concentration of oil. Hence putting into Nut Shell, in system V 1, composition B prepared with 2:1 ratio of S/CoS forms better SMEDDS compared to other two formulations and in all the cases oil concentration should be less than 20%. The systems V2 were prepared using Capmul MCM as oil, Labrasol as a surfactant and Transcutol P as a co-surfactant which produced three formulations A, B and C with varying ratios of S:CoS to 3:1, 2:1 and 1:1 respectively. Formulation V2 A (Fig (V2A)) created microemulsion region with oil up to 10% and S/CoS 80% but at larger oil concentrations it formed emulsion region having higher particle size which were not stable for longer time. Also the requirement of surfactant volume for single phase region in phase diagram was very high. Fig (V2B) and (V2C) showed comparatively smaller microemulsion region. So it can be concluded that excipients used for V2 are comparatively less suitable to form a SMEDDS then excipients of V1. It also suggests the comparatively less effectivity of Labrasol as a surfactant than Tween 80. The possible reason may the larger chain and greater solubilization capacity of Tween 80 than Labrasol. Also the combination of Labrasol with Transcutol P may not be able to form flexible, complex and easily reformable surface film. Thus it can be concluded that in system V2, composition A prepared with 3:1 ratio of S/CoS forms better microemulsion region compared to other two formulations and in all the cases oil concentration should be less than 10%. Next three compositions were prepared from third system V3 using Capmul MCM as oil, Tween 80 as surfactant and Transcutol P as co-surfactant with S/CoS ratio of 3:1, 2:1 and Akshay R. Koli 163

27 1:1 respectively. The V3 system has shown similar SMEDDS region as compared to system V1. It may be due to the presence of Tween 80 in the formulations. The Figure (V3 A, B and C) clarify that first two composition V3 A and composition V3 B formed self microemulsion region with up to 20% oil concentration where as third composition V3C did not show self-microemulsion region and stability of this microemulsion was poor. Composition V3 A (Fig (V3A)) was prepared with surfactant/co-surfactant (S/CoS) ratio of 3:1 which covers maximum microemulsion region as compare to other V3 compositions. As shown in Figure (V3A), the point where amount of oil is less than 15%, the water content is more than 80%. At this point microemulsion can be diluted to infinite which fulfills requirement of SMEDDS and also particle size of this microemulsion is less than 100nm (described in characterization of SMEDDS). The region where oil content is more than 15% and surfactant/cosurfactant is up to 60% also forms the microemulsion but these were found to be unstable on dilution. The phase diagram shows that when S/CoS reduces less than 40%, microemulsion region decreases drastically and coarse emulsion forms having particle greater than 100 nm. Hence it can be predicted that the concentration of S/CoS should be more than 40% to form self-microemulsion. Further, more amount of oil also entrap less water content and thereby results in coarse emulsion. Composition V1B was prepared using similar excipients but with S/CoS ratio of 2:1. In this system, after dilution amount of oil contained was limited up to 10%. At this point and below, microemulsion can be diluted to infinite which fulfills requirement of SMEDDS and also particle size of this microemulsion is less than 100nm (described in characterization of SMEDDS). Above these concentrations coarse emulsion formed. The third composition V3C was prepared using S/CoS as 1:1. Composition V3 C covers minimum microemulsion region compared to V3 A and B. It involves formation of microemulsion which is unstable on dilution after 20% oil (Fig (V3C)). Initially it formed self microemulsion but later on converted to emulsion as it moved towards higher concentration of oil. Hence putting into Nut Shell, in V3, composition A prepared with 3:1 ratio of S/CoS forms better SMEDDS compared to other two compositions and in all the cases oil concentration should be less than 10%. Akshay R. Koli 164

28 The Microemulsion region of V1 was higher than V3 in spite of having Tween 80 as surfactant in both systems. The reason may be the different co-surfactants compositions used in the compositions could have attributed to the difference in area of microemulsion as in the case of V1 where PEG 400 was used while in V2 and V3 it was Transcutol P. 5.4 Effect of Drug loading on the phase diagrams of the selected systems The incorporation of drug has considerable influence on the phase behavior of the spontaneously emulsifying systems. It has been reported that drug incorporation into microemulsion can affect the microemulsion region in phase diagram [18]. This can be due to drug penetration into the surfactant monolayer producing perturbations at the interface [18, 19].To verify this, the drugs were incorporated in to the selected oil:surfactant/cosurfactant system for Felodipine microemulsion and Valsartan SMEDDS and the area of the one phase region was observed and compared with the area of one phase region without drug Felodipine Microemulsion: To verify the effect of drug loading on one phase region of the phase diagram, 20 mg/ml (as per dose: 40mg/2ml) Felodipine was incorporated to Capmul MCM: Surfactant mixture (Tween 20:PEG 400= 2:1) and studied for microemulsion region in phase diagram by water titration. Akshay R. Koli 165

29 Figure : Pseudoternary phase diagram of Capmul MCM, Tween 20 and PEG 400 (Placebo) Figure : Pseudoternary phase diagram of Felodipine, Capmul MCM, Tween 20 and PEG 400 The phase diagrams indicating effect of Felodipine on phase behavior and area of microemulsion existence are shown in figure and It was expected that Felodipine would influence the phase behavior and the area of microemulsion formation Akshay R. Koli 166

30 as in these formula, Felodipine was present in 20mg/ml. Phase diagrams studies indicated that there was no difference observed in microemulsion region in the phase diagram between the drug loaded and placebo composition. This suggests that the presence of Felodipine does not affect the microemulsifying property of the composition Valsartan SMEDDS Similar results were found when 40mg/ml (as per dose: 80mg/2ml) of Valsartan was incorporated (10%w/w) to Capmul MCM: Surfactant mixture (Tween 80:PEG 400 = 3:1) system and studied for microemulsion region in phase diagram by water titration. A slight difference in microemulsion region in the phase diagram was observed between the drug loaded and placebo SMEDDS mixtures. The results are shown in Figure and for placebo SMEDDS and Valsartan loaded SMEDDS. It was expected that Valsartan would influence the phase behavior and the area of microemulsion formation. Phase diagrams studies indicated that there was slight influence of Valsartan on the area of microemulsion formation of the Capmul:Tween80:PEG 400 based system. Incorporation of Valsartan in system led to a slight reduction in the area of microemulsion formation of SMEDDS in Figure when compared to the area in Fig Valsartan, due to its low aqueous solubility and high surfactant mixture solubility, is likely to participate in the microemulsion by orienting at the interface. The reduction in the area of microemulsion formation could be due to Valsartan influenced interaction of surfactant and co-surfactant with oil. Akshay R. Koli 167

31 Figure : Pseudoternary phase diagram of Capmul MCM, Tween 80 and PEG 400 (Placebo) Figure : Pseudoternary phase diagram of Valsartan, Capmul MCM, Tween 80 and PEG 400 Akshay R. Koli 168

32 5.5 Preparation of Drug Loaded Microemulsions and SMEDDS Microemulsion and SMEDDS were prepared using the same method. However, the only difference was that in preparation of SMEDDs, addition of water was not done as in microemulsion. This SMEDDS is also known as microemulsion pre-concentrate because when this SMEDDS come in contact with water it will convert into microemulsion spontaneously. The flow chart for the preparation of drug loaded microemulsion is shown below: Fixed calculated quantity of oil, surfactant, co-surfactant & drug in completely dry beaker was taken. The drug was dissolved completely at room temperature under constant stirring on the magnetic stirrer. SMEDDS The required quantity of water was added drop wise with stirring. Allowed to form a clear and transparent liquid. Microemulsion Figure : Flow Chart of preparation of SMEDDS and Microemulsion Akshay R. Koli 169

33 5.5.1 Felodipine Microemulsion: A series of formulations were prepared with varying ratios of oil, surfactant and cosurfactant. Formulations F1 F9 were prepared using Capmul MCM as oil, Tween 20 as surfactant and PEG 400 as co-surfactant to optimize the concentration of oil and S mix, For microemulsion system, three different oil concentrations i.e 5%, 10% and 15% and three different concentrations of S mix i.e 40%, 45% and 50% were used. These concentrations were selected based on preliminary studies and pseudoternary phase diagram i.e. above 15% oil concentration, turbidity occurred and upto 50% S mix was sufficient to make clear microemulsion. The compositions are shown in Table Table : Compositions of Felodipine Microemulsion Systems (Batch F1 F9) Batch no. Felodipine (mg/ml) Oil %v/v S mix (2:1) % v/v F F F F F F F F F In all the formulations, the level of Felodipine was kept constant (i.e. 20 mg/ml of Felodipine). Briefly, oil, surfactant and co-surfactant were accurately weighed into glass vials according to their ratios. The Felodipine (20 mg/ml) was added in the mixture. Akshay R. Koli 170

34 Then, the components were mixed by gentle stirring and vortex mixing until Felodipine was completely dissolved. The mixture was stored at room temperature until used. So, prepared concentrate of microemulsion was composed of oil, surfactant, co-surfactant and drug. Water was added to microemulsion concentrate to make up the volume up to 100. The compositions which were optically clear have been evaluated further by constructing phase diagrams Valsartan SMEDDS: A series of formulations were prepared with varying ratios of oil, surfactant and cosurfactant. Formulations V1 (Table ) were prepared using Capmul MCM as oil, Tween 80 as surfactant and PEG 400 as cosurfactant. Similarly formulations V2 (Table ) were prepared with Capmul MCM as oil, Labrasol as surfactant and Transcutol P as cosurfactant. Third system containing formulations V3 (Table ) were prepared using combination of Capmul MCM, Tween 80 and Transcutol P as an oil, surfactant and co-surfactant respectively. In each system three formulations were prepared by varying ratio of Oil in three levels i.e 5%, 7.5% and 10% v/v with the optimized ratio of Surfactant mixture (S:CoS) as per the pseudo ternary phase diagram study (Section 5.3). For Tween 80: PEG 400 system (V1), highest one phase region was found in 2:1 ratio by pseudoternary phase diagram and hence selected as further preparation. For Labrasol: Transcutol P system (V2), highest one phase region was found in 3:1 ratio and hence selected for further preparation. For Tween 80: Transcutol P (V3) system, highest one phase region was found in 3:1 ratio and hence selected for further preparation. Formulations A, B and C were prepared by taking the concentration of oil as 5%, 7.5% and 10%. In each formulation concentration of valsartan was kept constant to 40 mg/ml. The volume of Valsartan SMEDDS was kept 2ml. The concentrations of oil, surfactant, and cosurfactant for Valsartan SMEDDS are recorded in Table (V1), Table (V2) and Table (V3). Akshay R. Koli 171

35 Table : Compositions of Valsartan SMEDDS 1 (V1) Ingredients (% v/v) A B C Valsartan (mg/ml) Capmul MCM Tween PEG Oil- Capmul MCM, Surfactant- Tween 80, Co-surfactant- PEG 400 (S:CoS=2:1) Table : Compositions of Valsartan SMEDDS 2 (V2) Vehicle (% v/v) A B C Valsartan (mg/ml) Capmul MCM Labrasol Transcutol P Oil- Capmul MCM, Surfactant- Labrasol, Co-surfactant- Transcutol P (S:CoS=3:1) Table : Compositions of Valsartan SMEDDS 3 (V3) Vehicle (% v/v) A B C Valsartan (mg/ml) Capmul MCM Tween Transcutol P Oil- Capmul MCM, Surfactant- Tween 80, Co-surfactant- Transcutol P (S:CoS=3:1) Akshay R. Koli 172

36 All the prepared compositions of Felodipine Microemulsion (Batches F1 F9) and Valsartan SMEDDS (Batches V1 A,B,C / V2 A,B,C / V3 A,B,C) were further carried forward for characterization and optimization in chapter 6. Akshay R. Koli 173

37 5.6 References 1. Talegaonkar, S., et al., Microemulsions: a novel approach to enhanced drug delivery. Recent Patents on Drug Delivery Formulation, (3): p Narang, A.S., D. Delmarre, and D. Gao, Stable drug encapsulation in micelles and microemulsions. International journal of pharmaceutics, (1-2): p Kawakami, K., et al., Microemulsion formulation for enhanced absorption of poorly soluble drugs: I. Prescription design. Journal of controlled release, (1): p Pouton, C.W., Lipid formulations for oral administration of drugs: nonemulsifying, self-emulsifying and self-microemulsifying drug delivery systems. European journal of pharmaceutical sciences, : p. S93-S Gedil, F., et al., Quantitative determination of felodipine in pharmaceuticals by high pressure liquid chromatography and uv spectroscopy. Turkish J. Pharm. Sci, (2): p Yin, Y.M., et al., Docetaxel microemulsion for enhanced oral bioavailability: Preparation and in vitro and in vivo evaluation. Journal of Controlled Release, (2): p Gupta, K., A. Wadodkar, and S. Wadodkar, UV-Spectrophotometric methods for estimation of Valsartan in bulk and tablet dosage form. Inter J ChemTech Res, (2): p Hauss, D.J., Oral lipid-based formulations. Advanced drug delivery reviews, (7): p Hanyšová, L., et al., Stability of ramipril in the solvents of different ph. Journal of pharmaceutical and biomedical analysis, (5): p Bhole, P. and V. Patil, Enhancement of water solubility of felodipine by preparing solid dispersion using poly-ethylene glycol 6000 and poly-vinyl alcohol. Asian journal of pharmaceutics, (3): p Shafiq, S., et al., Development and bioavailability assessment of ramipril nanoemulsion formulation. European Journal of Pharmaceutics and Biopharmaceutics, (2): p Akshay R. Koli 174

38 12. Prajapati, H.N., D.M. Dalrymple, and A.T.M. Serajuddin, A Comparative Evaluation of Mono-, Di-and Triglyceride of Medium Chain Fatty Acids by Lipid/Surfactant/Water Phase Diagram, Solubility Determination and Dispersion Testing for Application in Pharmaceutical Dosage Form Development. Pharmaceutical research, 2011: p Liu, R., Water-insoluble drug formulation, CRC press, : p Piao, H.M., et al., Preparation and evaluation of fexofenadine microemulsions for intranasal delivery. International journal of pharmaceutics, (1): p Singh, A.K., et al., Oral bioavailability enhancement of exemestane from selfmicroemulsifying drug delivery system. AAPS PharmSciTech, (3): p Shafiq, S. and F. Shakeel, Enhanced stability of ramipril in nanoemulsion containing Cremophor-EL: A technical note. AAPS PharmSciTech, (4): p Constantinides, P.P., Lipid microemulsions for improving drug dissolution and oral absorption: physical and biopharmaceutical aspects. Pharmaceutical research, (11): p Malcolmson, C. and M.J. Lawrence, A comparison of the incorporation of model steroids into non ionic micellar and microemulsion systems. Journal of pharmacy and pharmacology, (2): p Cuiné, J.F., et al., Increasing the proportional content of surfactant (Cremophor EL) relative to lipid in self-emulsifying lipid-based formulations of danazol reduces oral bioavailability in beagle dogs. Pharmaceutical research, (4): p Lawrence, M.J. and G.D. Rees, Microemulsion-based media as novel drug delivery systems. Advanced drug delivery reviews, (1): p Neslihan Gursoy, R. and S. Benita, Self-emulsifying drug delivery systems for improved oral delivery of lipophilic drugs. Biomedicine & pharmacotherapy, (3): p Akshay R. Koli 175

39 23. Lawrence, M.J., Surfactant systems: microemulsions and vesicles as vehicles for drug delivery. European journal of drug metabolism and pharmacokinetics, (3): p Patel, A.R. and P.R. Vavia, Preparation and in vivo evaluation of selfmicroemulsifying drug delivery system containing fenofibrate. The AAPS journal, (3): p Pouton, C., Effects of the inclusion of a model drug on the performance of self emulsifying formulations. Journal of pharmacy and pharmacology, (S12): p. 12. Akshay R. Koli 176